1. Field of the Invention
This invention generally relates to empirical methods for determining the fat and/or oil content of various food or non-food products. More specifically the invention is concerned with procedures for determining the fat and/or oil content of various products based upon the turbidity, and hence the ability to disperse light, of fat-containing extracts of such products.
2. Description of the Prior Art
Many industries, for example those associated with food production, the production of non-food products from plant or animal sources and various segments of the petroleum industry have need of processes for determining the fat and/or oil content of various fat and/or oil-containing materials. For example, in the food and feed industries it is important to be able to quickly and inexpensively determine the fat and/or oil content of various products because such determinations are used for such varied purposes as nutritional labelling, grading, processing and payment. Such determinations are especially important to the meat, dairy, oilseed and animal feed industries.
Many industries associated with fat and/or oil-containing containing products, and especially the food manufacturing industry, also have found that it is not practical, or necessary, to rely upon only those exhaustive, but highly accurate methods used to determine the ultimate chemical composition of a given product. In other words, in many instances it is not necessary to break down a product into its separate chemical constituents and/or to determine its true physical properties. This is fortunate because highly accurate chemical breakdown tests are often based upon the completion of an entire series of complex, costly, time-consuming and/or hazardous steps which usually require precise chemical techniques carried out by highly skilled personnel. Nonetheless, many well known and widely used analytical tests used in industry in general, and the food industry in particular, have all these characteristics.
For example, one of the most common, and most successful methods for making a quantitative analysis for fat content has been the Roese-Gottlieb method. It deserves some description here in order to elucidate the nature and extent of its complexities. In applying this procedure to say a milk sample, one begins by applying to a test sample of 10 grams of milk, 1.5 ml. of ammonium hydroxide. Thereafter, a total of 95 ml. of chemicals is added in six separate transfers, with mixing and shaking after each transfer. More specifically, these chemicals comprise, in all, 15 ml. of 95% alcohol, 40 ml. of diethyl ether, and 40 ml. of petroleum ether. They are introduced, in steps, as follows: (1) addition to the sample to be tested of three different ingredients; (2) vigorous shaking for thirty seconds; (3) addition of another ingredient; (4) another thirty second period of shaking; (5) lapse of enough time for the solution to separate sufficiently for the portion in the upper part of the container to become relatively clear; (6) careful drawing off of the heavier or unclear portion cf the solution from the bottom of the container; (7) extraction of the clear liquid remaining in the container; (8) addition of two more ingredients to the clear liquid; (9) another thirty second period of shaking; (10) addition of another ingredient; (11) another thirty second period of shaking; (12) addition of another ingredient; (13) another thirty second period of shaking; (14 ) allowing the solution to stand again until the top portion is practically clear; (15) drawing off a clear fat solution into a flask or other storage vessel (if the complete removal of all fat is required, still one more extraction is advisable, moreover the fat solvent that is drained off in the second or third extraction must, of course, be added to the portion saved from the first extraction); (16) evaporating the total amount of fat solvent removed during the preceding steps; (17) drying the residue of fat remaining after the evaporation and (18) weighing the fat. Note also that this procedure is for liquid milk; the complexities for testing solid foods such as cheese are even more extensive.
Attempts to avoid the complexities of such tests and the skills required to perform them have resulted in the development of other less complex chemical procedures. For example, one alternative chemical procedure requires fewer steps; but it requires the use of a potentially hazardous substance, sulfuric acid. Yet another alternative procedure eliminates the need for the use of sulfuric acid, but it is very time consuming. Fortunately, however, it has often been found quite acceptable in industries such as the food processing industry to sacrifice high accuracy with respect to determining the exact chemical identity of a given food's chemical constituents for improvements in speed, cost, simplicity, reproductibility of results, etc., through the use of certain alternative testing procedures.
Such alternative tests are, almost without exception, empirical in nature. That is to say that such empirical methods do not evaluate a true physical content or property of a given material. Rather, empirical tests are designed to elicit a behavior or response from a material by placing the material in, or subject it to, a highly prescribed set of conditions and then measuring the material's response to those conditions. However, such a response must be reproducible and must not be interfered with by other, extraneous, variations. It also must be measurable to a reliability which can be correlated to a required degree of accuracy with the intrinsic property whose measurement has been avoided.
With regard to the need for such empirical analytical procedures in the food industry, testing usually focuses on certain considerations and ignores others. Such testing also naturally divides itself into certain categories. For example, in testing many foods one can simply ignore the presence of water, minerals and trace nutrient adjuncts such as vitamins, in order to concentrate on those nutrients ingested primarily for the purposes of "fueling" human beings and/or animals. Such nutrients are generally thought of as being comprised of three groups: (1) proteins, composed of polypeptides which are in turn composed of polymers of one or more alpha-amino acids, (2) carbohydrates, distinct structures of carbon with hydrogen and oxygen in the ratio of two atoms to one, and (3) fats and oils, composed principally of carbon, hydrogen and oxygen (fats and oils may also contain small amounts of other elements). In any case, fats and/or oils serve as the basic fuel of humans and animals (in contrast to quick energy sources), and have the virtue of being retained as reserves in the form of emulsions in the body for future use. Humans and animals convert surpluses of protein and carbohydrate into fats for future use as fuels.
Fats and oils are often roughly distinguished from each other to a large degree by their being in, respectively, a solid or a liquid state. They are often thought of as the collection of all other nutrients beside strictly structured proteins, sugars, and starches. It should be noted here, however, that for many food technology purposes and for the purposes of this patent disclosure, the terms "fat(s)" and "oil(s)" can be thought of as being synonymous. Chemically, they cover a vast variety of structural types: esters, lipids, glycols, steroids, glycerides, etc., and herein lies the source of many difficulties encountered in finding suitable analytical methods for their assay. Considering their chemical as well as their physical differences, it is probably not unduly pessimistic to suggest that a single procedure capable of selectively isolating and measuring each characteristic of a fat or an oil is no where on the food technology horizon. It best then, the food processing industry has usually sought out only those subtractive methods which have an ability to group as wide a variety of fats and oils as possible. Specific means are then found to remove or to otherwise ignore the effects of extraneous materials.
Some food technologies have taken a very different tack and abandoned the idea of a single, all embracing test method, and recognize, realistically, that no food material contains every kind of fat. For example, it is hardly necessary to provide for the presence of palmitins in fish, cholesterol in olive oil, stearins in corn, etc. Furthermore, in many applications the identification of the chemical character of fat content simply is not necessary. As a case in point, the heat of combustion (common food calories) of all fats and oils can be taken as 9 calories per gram, whether in butter, corn, oleo, or ham. Any error is usually within the error introduced by water content variation from sample to sample. Thus, it would seem that a practical approach for what might be called "control" testing of foods could be made bu first ascertaining the foods to be assayed and therefore the fats to be encountered. Second, an empirical test method need only be comprehensive for these materials. Finally, means of removal of major interferences may be sought. However, such removal is only necessary if it causes confusion in a control test, and if, in the correlation of results leading to the test calibration, then otherwise cannot be discounted or otherwise allowed for with sufficient accuracy. To these ends, the food industry has developed a number of empirical tests to determine the fat (and/or oil) content of various food products.
For example, U.S. Pat. No. 2,863,734 (the 734 patent) teaches a method for determining fat content (especially in fluid dairy products) by use of a detergent composed of two surface active agents. One of the surface active agents is an alcoholic, non-ionic agent which is employed to destabilize a fat emulsion. The 734 patent postulants that this action follows from the non-ionic agent's ability to solubilize protein-lecithin coatings surrounding fat globules and thereby destroying their stability as dispersed elements in an oil/water emulsion. The second surface active agent is an anionic agent whose function is to solubilize the non-fat material associated with the fat. Various anionic agents (e.g., dioctyl sodium phosphate) can be employed for this purpose. Measured quantities of the detergent composition are then added to measured quantities of a liquid dairy product sample. The mixture is also warmed to induce separation of a fat layer in the flask. A fat immiscible liquid is then added to displace the layer of fat into a graduated neck of the flask in order to measure the volume of said fat layer.
U.S. Pat. No. 3,062,623 ("the 623 patent") teaches a method for determining fat content, particularly in the context of blood serum. The process closely resembles the technology previously discussed with respect to the 734 patent. It also should be noted in passing that the 623 patent refers to the composition in the 734 patent as "a high molecular weight organic surface active agent having detergent properties". In any event, the 623 patent teaches use of the same general anionic surface active agents as those discussed in the 734 patent. It also teaches the use of another agent, ethoxy triglycol, to render the anionic surface active agent miscible in an aqueous medium. The 623 patent also suggests use of various alcohols, including methyl alcohol, as ingredients of the non-ionic agent compositions.
U.S. Pat. No. 3,351,431 teaches a fat content determination based upon use of a solvent comprising acetone, petroleum ether, and n-butyl alcohol. Essentially the process involves extraction of the fat with the solvent, physical separation of the fatty extract from the raffinate, evaporation of the solvent and weighing of the remaining dried fat.
U.S. Pat. No. 3,746,511 teaches a turbidimetric method for determining milk fat content. It employs acetic acid in combination with a class of surfactants having the general formula of certain quaternary halide salts: dialkyl, dimethyl ammonium halide, with the alkyl groups being the same. The turbidimetric aspect of this invention is based upon the formation of a colloidal dispersion of the milk fat which has a linear response of light absorbance in the 1.5 to 7 percent milk fat content range.
U.S. Pat. No. 3,960,493 teaches a nephelometric procedure which involves adding two distinct reagent solutions to a milk sample to form, sequentially and respectively, colloid dispersions of protein and fat, while effecting solubilization of the other. Nephelometric readings are taken after the addition of: (1) a protein reagent solution which is anhydrous (e.g., acetic anhydride, para-toluene sulfonic acid and acetic acid) and (2) a fat reagent solution, such as water with a small amount of non-ionic surfactant, whose function is to precipitate fat colloids.
U.S. Pat. No. 4,497,898 teaches a method of determining the fat content cf milk by a triple-beam spectrophotometer system wherein two of the beams measure transmittance of separate streams of a sample, i.e., a stream with a reagent for developing coloration intensity proportional to the protein level and a reference stream without the color developing agent. The third beam measures the intensity of a light source. Comparison of the relative transmittance of the two streams to the relative transmittance of beams passing through a baseline fluid such as water enables one to determine separately, but in the same fashion, the protein and the fat content of the sample.
It should also be noted that some prior art methods of determining fat content of a fat-containing sample also have employed tetrachloroethylene as a fat solvent. For example, the reference source: Official Methods of Analysis of the Association of Official Analytical Chemists ("AOAC"), protocol number 24.006 of the 14th Ed., 1984, teaches fat extraction from a meat sample by use of tetrachloroethylene as a fat solvent. In this particular process, the fat is extracted from the meat in the presence of a drying agent such as CaSO.sub.4. Thereafter, the fat extract is filtered and the specific gravity of the extract is correlated to the known fat content of samples.
The empirical processes disclosed in many of the above noted references usually employ phenomena associated with phase separation in liquids and/or phenomena associated with light-scattering. A few words with respect to each may be of some use here. With respect to phase separations, it is believed that an electrical double layer exists at the interface between the two phases of a colloidal dispersion. When the electric potential is measured between the so-called Guoy section of this double layer and the bulk dispersing phase, it is known as the "zeta potential". Its value is generally regarded as a partial indication of the stability of a colloidal system. It is probably not necessary to delve deeply into fundamental light-scattering theory, since many good reviews on the subject, in the context of food technology, are available (e.g., see Light Scattering by Milk Globules, by Walstra, Netherlands Milk Dairy Journal (19:93 (1965)). Suffice it to say that under certain conditions, turbid suspensions obey the Beer-Lambert law as applied to colored solutions. The attenuation of the light beam is due to scattering of light out of the direction of the incident beam rather than by molecular absorption.
With respect to the teachings of this patent disclosure, it should also be noted that it is well known that optical dispersion of a colloidal suspension of oil in an immiscible medium at a given wavelength is a function of the fat content in the suspension and thus of the fat content of the original sample. It is also known that since a visible absorption spectrum of such a suspension shows no peaks, the particular wavelength employed is arbitrary and may be chosen as convenient. However, those skilled in this art will appreciate that in any scattering and/or absorptiometric system, precautions have to be taken to eliminate reflection of scattered light back into the optical system. In addition, the use of an interference filler will insure maximum adherence to the Beer-Lambert law. It will also be appreciated that in colloidal systems of the type found in this patent disclosure, agglomeration will occur when the zeta potential is zero. Certain surfactants also are known to have an effect upon the zeta potential under certain conditions. Those skilled in this art also will appreciate that in an acidic solution, anionic and non-ionic surfactants have greatly reduced surface active properties, while cationic surfactants retain these properties.
With respect to the empirical use of such colloidal suspensions in general analytical chemistry, as well as in the context of this patent disclosure, it should also be noted that it is alwaYs desirable to use a stable, reproducible suspension in any measurement operation. This is especially true in automated continuous flow analysis where the optical surfaces of the flow cell must be kept as clean as possible in order to eliminate baseline drift. This pertains particularly to analysis of milk fats, where an unstable suspension rapidly produces a large buildup of aggregated colloidal particles throughout the glass part of the manifold, including the flowcell. Since agglomeration of colloidal particles also materially influences optical absorbance, this situation is to be avoided as much as possible.
In any event, each of the above noted techniques for determining the fat content of foods has certain virtues and drawbacks. The drawbacks usually revolve around the chemical complexity of the test, the accuracy achieved, the character of the ingredients and equipment and the time and skills needed to carry out the technique within prescribed degrees of precision. Hence any test which makes improvements in any of these areas of concern would be most welcome; this is especially true in the food processing industry.